The overall goal of this line of research is to gain a better understanding of the underlying mechanisms that determine successful nerve regeneration in the peripheral nervous system; this is a high priority research area for the VA healthcare mission. Following injury to a peripheral nerve the denervated distal nerve segment undergoes remarkable changes including loss of the blood-nerve barrier, Schwann cell proliferation, macrophage invasion, and the production of numerous cytokines and neurotrophic factors. The aggregate consequence of these changes is that the denervated nerve becomes a permissive and even preferred target for regenerating axons from the proximal nerve segment. However, it often surprises people that although peripheral nerves display robust axonal regeneration, only approximately 10% of adults will recover normal nerve function using state-of-the-art repair techniques. This success rate has not markedly improved since the introduction of enhanced microsurgical techniques utilizing modern surgical microscopes several decades ago. Clearly there is a need for new ideas and approaches to this problem in order to improve these outcome statistics. Previous work from several laboratories, including our own, has shown that earlier reinnervation of an end-organ target such as muscle results in a greater overall level of functional recovery. Thus a major key to improving functional recovery will be to decrease the amount of time that distal end-organ targets such as muscle remain denervated. Another factor that will improve functional recovery following nerve repair will be to increase the extent of accurate reinnervation of origina end-organ targets. The possible role that the denervated end-organ target itself (e.g. muscle) may play in these phenomena during the regeneration period is largely unexplored. The work proposed in this application will begin to address this question using model systems in the mouse to assess both the extent and accuracy of motor neuron regeneration at the level of the terminal nerve branch. Exosomes (nanometer sized extracellular vesicles) are secreted by just about every cell type that has been examined, and it has recently been discovered that they become a protected environment for various forms of RNA and proteins and thus can function as novel long-distance messengers. Our working hypothesis is that muscle, as an end-organ, elaborates signals that travel within the denervated nerve and that these signals influence the speed and accuracy of regenerating axons. We further suggest that exosomes produced by muscle may be one such mechanism by which such signals travel within the denervated nerve. In support of this hypothesis we present in vivo data that demonstrates that the accuracy of regenerating motor neurons is dependent upon the denervated distal nerve segment remaining in uninterrupted continuity with muscle. We also present preliminary data that exosomes harvested from a muscle cell line and applied to a denervated nerve in vivo results in significantly greater and faster axonal regeneration in two different regeneration models. The experiments proposed in this application could provide a paradigm-shift in our approach to peripheral nerve repair by focusing on the role of exosomes produced by target end-organs. This innovative idea is a significant addition to existing interventions that directly target the motor neuron itself, and seeks to impact clinical and research attention by highlighting the distal denervated nerve as a general delivery device for novel therapeutics.